Cholera is an acute water-borne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. Most studies on V. cholerae infection have focused on identifying factors that promote initial adherence and colonization of the intestinal mucosa and enterotoxicity. However, fewer studies have focused on those factors that promote detachment of Vibrios, their dissemination throughout the small intestine and return to the aquatic environment. Expression of hemagglutinin (HA)/protease and motility have been suggested to facilitate V. cholerae detachment from the intestinal mucosa when infective Vibrios reach high cell density. Studies conducted in our laboratory have revealed a complex interplay between the second messenger cyclic diguanylate (c-di-GMP), the general stress response regulator RpoS and quorum sensing in the coordinate regulation of HA/protease and motility. In addition, we have examined the role of RpoS and the histone-like nucleoid structuring protein (H-NS) in the regulation of motility. Specifically, we have demonstrated that (i) c-di-GMP negatively regulates HA/protease expression, (ii) RpoS diminishes the c-di-GMP pool to enhance HA/protease and motility, (iii) c-di-GMP negatively regulates RpoS expression when cells are not in quorum sensing mode, and (iv) RpoS enhances motility by both lowering the c-di-GMP pool and attenuating H-NS repression of the motility regulator FlrA. Here we propose to thoroughly characterize these new regulatory pathways and how they affect environmental stress response and detachment of V. cholerae from intestinal cells. In Aim 1 we will determine the signaling pathway connecting c-di-GMP to RpoS expression and investigate the role of quorum sensing in this regulation. Then, we will examine if the negative regulation of RpoS by c-di-GMP diminishes the capacity of V. cholerae to withstand environmental stresses. In Aim 2 we will determine the regulatory events involved in RpoS and H-NS regulation of motility. Briefly, we will determine the c-di- GMP metabolic enzymes regulated by RpoS and combine microbial genetics, DNA binding assays and chromatin immunoprecipitation (ChIP) to determine how expression of RpoS diminishes H-NS occupancy at the flrA promoter. Finally, we will exploit the yeast two-hybrid system and pull down assays to determine the molecular basis for the flagellated non-motile (Mot-) phenotype exhibited by hns mutants testing the hypothesis that H-NS directly interacts with the flagellar motor. In Aim 3 we will determine the role of c-di-GMP in V. cholerae detachment from the intestinal mucosa. To this end, we will exploit the use of strains in which the c-di-GMP pool can be artificially increased and examine the effect of this second messenger in V. cholerae adherence and detachment from intestinal cells using in vitro and in vivo models of infection. As an in vitro model we will use HT29-18N2 cultured cells which can be induced to differentiate into mucin-secreting goblet cells. As an in vivo model we will use adult rabbit ileal loops.